The trends in modern plant extract technology are moving towards the use of a greater variety of raw materials, a more complete utilisation of these raw materials, a speeding up of processes and a presentation of a greater variety of bases and finished products. Some of these developments are made possible through improvements of existing processes and process equipments, as well as through introduction of new processes and equipments. The use of enzyme preparations as processing aids play a key role in these developments.
During fruit juice manufacture enzyme preparations are often used in the steps of extraction and liquefaction of fruit and fruit juice clarification. The commercial enzyme preparations contain a mixture of enzymes which degrade the pectin polymers (including pectin lyases, polygalacturonases, pectin esterases, galactanases, arabinases), as well as other enzymes like cellulases and xylanases.
New developments are going into the direction of cloudy juices and extracts from plant material. The mechanism why some of these extracts stay cloudy while others spontaneously clarify is not well understood. Therefore emphasis is put into research to learn more about the background of cloudiness and cloud stability.
Pectins (pectin-polymers) occur in nature as constituents of higher plant cell walls. They are found in primary cell wall and middle lamella where they are embedded in cellulose fibrils. The composition of pectin is variable among plant species and moreover dependent on the age and the maturity of the fruit. Among the richest sources of pectins are lemons and oranges, which can contain up to 30% of this polysaccharide.
Most pectin-polymers are composed of smooth regions, i.e. linear homogalacturonan, and hairy (ramified) regions.
The linear homogalacturonan is composed of chains of 1,4-linked .alpha.-D-galacturonic acid, the polygalacturonic acid is methoxylated to a varying extent, and may further be partially acetylated. The linear homogalacturonan can be degraded and depolymerized by different enzymes: Pectin lyase which cleaves the galacturonosyl bonds of highly methoxylated pectins by .beta.-elimination. Pectate lyase cleaves galacturonosyl bonds in the non-methoxylated parts of pectin by .beta.-elimination, and poly-galacturonase hydrolyses the glycosidic linkages in the non-methoxylated part of homogalacturonan. The action of pectate lyase and polygalacturonase is facilitated by pectin methylesterase which catalyses the removal of methanol from homogalacturonan, resulting in the formation of pectic acid (polygalacturonic acid). Enzymes or enzyme combinations which have the ability to depolymerize homogalacturonan are designated homogalacturonan depolymerizing enzymes in the following disclosure.
The hairy regions consist of a rhamnogalacturonan backbone with side-branches of varying length. The pectin hairy regions might be heterogeneous, with regions with extensive branching, regions with less extensive branching, and regions where the backbone is rich in galacturonic acid with an extensive branching with .beta.-linked xylose (xylogalacturonan).
The composition of the very complex structure of the hairy regions vary according to the source of the plant cell wall, cf. Schols et al. in Carbohydrate Research 206, 1990, pp. 117-129; O'Neill et al. in "Methods in Plant Biochemistry", Vol. 2, Carbohydrates, P. M. Dey (Ed.), 1990, Academic Press, London, pp. 415-441; Voragen and Schols in "Structural Studies of Plant Cell-Wall Polysaccharides Using Enzymes", Special Publication No. 134, The Royal Society of Chemistry 1993 and Carpita and Gibeaut in The Plant Journal 3(1), 1993, pp. 1-30.
Rhamnogalacturonans are polysaccharides with more or less regularly alternating rhamnose and galacturonic acid residues in the backbone. The rhamnogalacturonan backbone in the hairy regions has acetyl groups on the galacturonic acid residues (cf. H. A. Schols in Carbohydrate Research 206, 1990, pp.117-129).
The degradation of the backbone of the hairy regions is performed by enzymes designated rhamnogalacturonases (RGases). RGases are believed to hydrolyse the bond between rhamnose and galacturonic acid. In order to facilitate the activity of RGases it may be desirable to reduce the degree of acetylation of the backbone, e.g., by use of the enzyme rhamnogalacturonan acetyl esterase (cf. Searle-van Leeuwen et al. in Appl. Microbiol. Biotech. 38, 1992, p. 347-349). Furthermore, a reduced degree of branching of parts of the hairy regions may facilitate the activity of rhamnogalacturonanases. The reduced degree of branching may be obtained by enzymes which attack the side-branches.
The isolation and purification of a RGase from Aspergillus aculeatus are described by Schols et al. in Carbohydrate Research 206, 1990, p. 105-115. Another type of RGase from A. aculeatus is described in WO 92/19728.
Enzymes which attack the backbone of hairy regions are defined in the following disclosure as any enzyme or combination of enzymes which has the capability of attacking (by hydrolysis, .beta.-eliminations, or otherwise) the backbone of hairy regions, e.g., rhamnogalacturonase containing enzymes preparations.
The side-branches include monosaccharides like xylose, galactose and arabinose, and oligo and polysaccharides like arabinan, galactan and arabinogalactan.
Galactan contains .beta.-1,4 linked galactose in the backbone. Galactans are degraded by .beta.-1,4-galactanases (EC 3.2.1.89) (in short galactanases). Reference can be made to R. F. H. Dekker and G. N. Richards, "Hemicellulases, their Occurence, Purification, Properties and Mode of Action"in R. S. Tipson and D. Horton, Advances in Carbohydrate Chemistry and Biochemistry, Academic Press 32, 277-352 (1976); R. F. H. Dekker, "The Hemicellulase Group of Enzymes", in J. M. V. Blanchard and J. R. Mitchell, Polysaccharides in Food, Butterworths, 93-108 (1979), and A. G. J. Voragen, F. Geerst and W. Pilnik "Hemicellulases in Enzymatic Fruit Processing", in P. Depuy, Use of Enzymes in Food Technology, Technique et Documentation Lavoisier, 497-502 (1982). One example of a galactanase is the galactanase described in WO 92/13945.
Further, galactan and galactose sidebranches are degraded by the exo-acting enzyme .beta.-galactosidase.
Galactans might have arabinose sidebranches (arabinogalactan), these sidebranches are hydrolysed by .alpha.-arabinosidase. The partial or full removal of the arabinose sidebranches might facilitate the activity of galactanases.
Arabinan is composed of a backbone of .alpha.-L-arabinose subunits linked .alpha.-(-&gt;5) to each other and side chains linked .alpha.-(1-&gt;3) or .alpha.-(1-&gt;2) to the backbone. Enzymes which are capable of degrading arabinan backbone are designated arabinanases. The sidebranches of arabinan can be hydrolysed by .alpha.-arabinosidases (Rombouts et al., Carbohydrate Polymers 9, 1988, p. 25), which can also hydrolyse linear arabinan from the non-reducing end.
Xylose side-branches can be removed by .beta.-xylosidase.
Enzymes which attack the hairs of the hairy regions of pectin will in the following disclosure include all enzymes which have the ability to degrade fully or partially the hairs of the hairy regions of pectin, including galactanase, .beta.-galactosidase, .beta.-xylosidase, arabinanase and .alpha.-arabinosidase, or any combination hereof.
The term hairy region degrading enzymes are meant to include both enzymes which attack the hair or the backbone of the hairy regions.
The enzyme preparations used for production of plant extract like juice contain both homogalacturonan depolymerization enzymes as well as other enzymes like enzymes which attack the hairy regions of pectin and/or cellulose. Such enzyme combinations often lead to extracts with a low cloudiness and/or a low cloud stability. Homogalacturonan depolymerization activity can be measured as PSU activity (Analytical method obtainable from Novo Nordisk A/S as AF-269).